A study on prevalence and clinico-mycological profile of superficial fungal infections in a tertiary care hospital

The incidence of superficial mycoses has increased recently due to frequent usage of antibiotics and various immunosuppressive conditions. In the background of immunosuppression, detection of these agents becomes necessary for effective management and prevention of further invasions. The objective of the study was to determine the prevalence and causative agents of superficial mycoses in order to provide early and efficient treatment. A cross-sectional study was conducted for 3 months (JuneAugust 2015) on patients with suspected superficial mycoses attending our Dermatology OPD. Skin, hair and nail samples were collected and transported aseptically to Microbiology laboratory for further processing. Out of the 394 cases that attended the dermatology OPD, 48 clinically suspected cases of superficial mycoses were studied. Prevalence of superficial mycoses was 10.4%. 41 cases showed positive culture, while only 37 cases were KOH positive. T. rubrum was the most common dermatophyte (48.27%) and the commonest causative agent (34.14%) while Candida spp were the most common non-dermatophyte (50%) and 3rd most common etiological agent (12.20%) isolated. We concluded that along with dermatophytes, dermatomycotic fungi are also emerging as an important cause of superficial mycoses. Though culture was found to be more sensitive than KOH mount, both techniques are important tools of diagnosis.

Original Research Article https://doi.org/10.20546/ijcmas.2019.801.268

A Study on Prevalence and Clinico-Mycological Profile of Superficial

Fungal Infections in a Tertiary Care Hospital C.L Vasudha 1 *, B Anuradha 2 and Meer Muzaffar Ali Faizan 3

1

Department of Microbiology, Mamata Medical College, Rotary nagar, Khammam,

Telangana – 507002, India

2

Dept of Microbiology, Mamata Medical College, Khammam, Telangana, India

3

Intern, Mamata Medical College, Khammam, Telangana, India

*Corresponding author

A B S T R A C T

Introduction

Fungal infections are worldwide in

distribution of which superficial infections are

the most common human infections (Brown et

al., 2012) These are rarely life threatening but

they have their own negative effects on the

patient’s emotional, social and occupational

status (Langan et al., 2010) In the tropical and subtropical countries like India superficial fungal infections are more prevalent due to the fact that heat and moist conditions are more

suitable for the growth of fungi (Peerapur et

al., 2004) Superficial mycoses refer to the diseases of the skin and its appendages caused

by fungi caused by Dermatophytes, candida

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 01 (2019)

Journal homepage: http://www.ijcmas.com

The incidence of superficial mycoses has increased recently due to frequent usage of antibiotics and various immunosuppressive conditions In the background of immunosuppression, detection of these agents becomes necessary for effective management and prevention of further invasions The objective of the study was to determine the prevalence and causative agents of superficial mycoses in order to provide early and efficient treatment A cross-sectional study was conducted for 3 months (June-August 2015) on patients with suspected superficial mycoses attending our Dermatology OPD Skin, hair and nail samples were collected and transported aseptically to Microbiology laboratory for further processing Out of the 394 cases that attended the dermatology OPD, 48 clinically suspected cases of superficial mycoses were studied Prevalence of superficial mycoses was 10.4% 41 cases showed positive culture, while

only 37 cases were KOH positive T rubrum was the most common dermatophyte (48.27%) and the commonest causative agent (34.14%) while Candida spp were the most

common non-dermatophyte (50%) and 3rd most common etiological agent (12.20%) isolated We concluded that along with dermatophytes, dermatomycotic fungi are also emerging as an important cause of superficial mycoses Though culture was found to be more sensitive than KOH mount, both techniques are important tools of diagnosis

K e y w o r d s

Superficial

mycoses,

Dermatophytes,

Tinea corporis,

Onychomycosis,

T rubrum, Candida

spp

Accepted:

18 December 2018

Available Online:

10 January 2019

Article Info

species and other non-dermatophytic moulds

such as Aspergillus spp, Fusarium sppand

Acremonium species They have affinity for

tissues that are keratin rich like skin, hair and

nails, and thus they produce inflammatory

response associated with clinical signs and

symptoms such as itching etc, along with

cosmetic defects The three genera of

dermatophytes that are recognised are

Trichophyton (Grover and Roy, 2003)

Over the last decades, an increasing number of

non – dermatophyte filamentous fungi have

been recognized as agents of skin and nail

infections in humans, producing lesions

clinically similar to those caused by

dermatophytes (Patel et al., 2010)

Recently there has been an increase in the

incidence of fungal infections This increase

may be a result of frequent usage of

antibiotics, immunosuppressive drugs and

various conditions like organtransplantations,

Immunodeficiency Virus (HIV) infections

(Petmy et al., 2004)

Previously these infections were considered as

mere cosmetic problems but in recent years

these have gained importance as major public

health problems This is because affected

patients experience embarrassment in social

and work situations, where they feel unclean,

unwilling to allow their hands or feet to be

seen Patients may fear that they will transmit

their infection to family members, friends, or

co-workers, fears that can lead to diminished

self-esteem and the avoidance of close

relationship In fact, many patients with fungal

nail infections experience serious physical,

psychosocial, and occupational effects as a

result of this disease

A correct diagnosis is important to initiate

appropriate treatment and also essential for

epidemiological purposes In the background

of immunosuppression, detection of these agents becomes mandatory for the effective management of mycoses to prevent further

invasions (Kannan et al., 2006)

The present study was conducted to know the prevalence and possible etiological agents of superficial fungal infections in our area, in order to provide early and appropriate treatment to reduce the social burden

The objective of the study is to determine the prevalence and causative agents of superficial fungal infections in order to provide early and efficient treatment and reduce the morbidity

Materials and Methods

This is a cross-sectional study conducted for a period of 3 months (June- August 2015) and included patients of various age groups with suspected superficial mycoses attending the outpatient department (OPD) of Dermatology

of our hospital The institutional ethical committee clearance was obtained

Samples such as skin scrapings, nail clippings, subungual scrapings and hair were collected under aseptic conditions after obtaining informed consent from the patients

Specimen collection (Larone, 2011)

Scrapings of skin were taken from the active, peripheral edge of the lesion with a scalpel or the end of microscope slide, after it had been cleansed with 70% alcohol Hair was plucked out from the root using sterile forceps Brushings were taken from the area of scaly scalp Infected nails were cleansed with an alcohol wipe and then scraped deeply enough (subungual scrapings) to obtain recently invaded nail tissue, using blunt end of the scalpel The initial scrapings were discarded

as they are usually contaminated The samples

so collected were transported in a sterile

container or a black paper envelope to

Microbiology laboratory for further

processing

Specimen processing (Larone, 2011)

The samples received in the Microbiology

laboratory were processed as follows:

Microscopy/ Potassium hydroxide (KOH)

wet mount preparation

Portion of sample is placed on a labelled slide

to which few drops of 10% KOH (for skin

&hair samples) solution is added Cover slip is

placed over it and the slide is gently heated

over flame without boiling The slide is then

carefully examined microscopically to detect

presence of fungal elements The nail samples

were submerged in 20% KOH (Flores JM et

al., 2009) overnight for complete softening

and clearing, in order to afford good visibility

Culture

Media used for fungal culture were

Sabouraud’s Dextrose Agar (SDA) with

antibiotics and cycloheximide (for

dermatophytes) and plain SDA without

cycloheximide (for candida and

non-dermatophyte) Samples were inoculated on

both the media, incubated at 300Cand

examined daily for growth

Confirmation of isolates

The growth of moulds was confirmed by

Colony characteristics

Tease Mount with Lactophenol Cotton Blue

(LPCB) to detect the morphology of fungus

Slide culture technique

The growth of yeasts was confirmed by

Colony characteristics

Gram stain of the colony

Germ tube test Chrom agar

Tease mount (Larone, 2011)

Place a drop of LPCB on a clean glass slide With a sterile bent dissecting needle or sterile loop, remove a small portion of the colony from the agar surface and place it in the drop

of LPCB With two dissecting needles, gently tease apart the mycelial mass of the colony on the slide, cover with a coverslip, and observe under the microscope with low power (10X) and high-dry (40X) objective lenses

Slide culture technique (Larone, 2011)

It is done to study the undisturbed morphology

of fungi which helps to identify the fungal species A microscopic slide is placed on a bent glass rod at the bottom of the petri dish with a filter paper A piece of 1 cm block of SDA is put on the slide The fungal growth obtained is inoculated at four sides of the agar block and covered with a sterile cover slip Few drops of water are added on the filter paper to avoid drying of the agar The lid of the petri dish is closed, the preparation is left

at room temperature When the growth appears approximately after 7-14 days (dermatophytes) or 3-4 days (non-dermatophytic molds) a drop of lactophenol cotton blue (LCB) is placed on the slide and the cover slip from the block is placed on it This slide is examined microscopically for the structural arrangement of the fungi

Gram stain (Allen et al., 2005)

Smear from the yeast-like colonies were prepared on a clean glass slide and subjected

to Gram stain (according to standard procedure) They were then observed under microscope for the presence of Gram positive budding yeast-like cells

Germ tube test (Larone, 2011)

The culture showing yeast like dry and pasty

colonies are treated with mammalian (foetal,

bovine, sheep or normal human) serum and

incubated at 370C for 2 to 4hours A drop of

this suspension is placed on the slide, covered

microscopically for germ tubes which are seen

as long tube like projections extending from

the yeast cells

Chrom agar (Chander, 2009)

It is selective and differential chromogenic

medium used for identification of various

candida species It is based on direct detection

of specific enzymatic activities by adding

multiple chemical dyes i.e substrates of

fluorochrome to media Yeast like colonies

obtained from SDA are inoculated on this

agar, incubated at room temperature (300C)

for 48-72 hours and looked for following

colours of colonies:-

C.albicans -Light green

C.dubliniensis - Dark green

C.glabrata - Pink to Purple

C.krusei - Pink

C.parapsilosis - Cream to Pale pink

C.tropicalis - Blue with Pink halo

All the materials required for culture were

obtained from HiMedia Laboratories Pvt Ltd,

Mumbai, India

Statistical analysis

The results were expressed as percentages for

the analysis of various data Microsoft excel

was used for the interpretation of these results

Results and Discussion

A total of 394 patients attended the

dermatology OPD during the study period

Out of these, 48 clinically suspected cases of superficial fungal infections were studied Out

of the 48 clinical cases 62.5% (30) were males and 37.5% (18) were females with a male to female ratio of 1.67:1 Prevalence of superficial fungal infections in our study was 10.4% (41/394) The most common age group affected was between 31-45 years (33.33%), followed by 16-30 years (27.08%) (Table 1) Out of the 48 clinical samples, 39.58% (19/48) were skin scrapings, 31.25% (15/48) were hair samples and 29.16% (14/48) were nail clippings [Chart 1] Out of the 48 cases, 85.41% (41) were culture positive, of which 77.08% (37) were both KOH and culture positive, 8.33% (4) were KOH negative and culture positive 14.58% (7) were both KOH and culture negative There were no cases where KOH was positive and culture negative (Table 2)

Out of the 41 positive cultures obtained, 70.73% (29) were dermatophytes and 29.26% (12) were non-dermatophytes Among the

dermatophytes, T.rubrum 48.27% (14/29) was

the most common isolate and among

non-dermatophytes candida species 50% (6/12)

were the commonest isolates

Overall, T.rubrum 34.14% (14/41) was the

T.mentagrophytes 19.51% (8/41), C.albicans 12.20% (5/41) and M.gypseum 9.76% (4/41) Other isolates were T.verrucosum, Curvularia, A.fumigatus (4.89% each, i.e 2 each) and T.tonsurans, C.parapsilosis, Bipolaris and Alternaria spp (22.43% each i.e 1 each)

[Chart 2]

There were no cases of mixed infections,

Malassezia infections and infections due to Epidermophyton spp in this study

Different types of Superficial mycoses cases which were studied are described in Table 3 It was observed that Onychomycoses (29.17%),

Tinea corporis (25%) and Tinea capitis

(20.83%) were the commonest types of

Superficial mycoses in our study (Fig 1 and

2)

Significance of the results related to

research work

Superficial mycoses form a large group of

patients attending the Dermatology OPD of

our tertiary care hospital Apart from the

clinical symptoms superficial fungal infections

can cause debilitating effects on a person’s

quality of life Although rarely life threatening

they may in some circumstances spread to

other individuals or become invasive Most

superficial fungal infections are easily

diagnosed and readily amenable to treatment

(Abida Malik et al., 2014)

Prevalence of superficial fungal infections in

our study was 10.4% This was in

concordance with other studies which showed

similar prevalence rate of 12.61% (Flores et

al., 2009) and 9.16% (Eftekharjo et al., 2015)

In the present study, males were more affected

than female with a male to female ratio of

1.67:1 Other studies done in India, showed

similar observation with male to female ratios

of 1.79:1, 4.26:1, 1.8:1 and 1.63:1 respectively

(Grover et al., 2003; Kannan et al., 2006;

Nawal et al., 2012 and Surendran et al., 2014)

But a study done by Dulla et al., 2015, showed

that females were more affected than males

with female to male ratio of 1.1:1 Increased

incidence in males can be attributed to their

greater outdoor exposure and physical activity

Persons of all ages were susceptible but

maximum cases of fungal infection occurred

between the age group of 31-45 years

(33.33%) followed by 16-30 years (27.08%)

This was in concordance with study done by

Dulla et al., (2015), which showed a higher

prevalence in the age group 31-40 (26.4%)

years

The culture positivity rate was 85.41%, which was in concordance with study done in Iran

(Eftekharjo et al., 2015), which showed 84.1%

culture positivity But other studies (Surendran

et al., 2014; Dulla et al., 2015 and Prasad et al., 2013) done in India showed lesser culture

positivity rate of 39%, 57.6% and 69.51% respectively

The KOH positivity rate was 77.08%, and comparable with studies done in various parts

of India 70.4% (Dulla et al., 2015) and 75.57% (Prasad et al., 2013) But lesser when compared to the study done by Surendran et al., (2014) which showed a positivity of 96%

In our study, 8.33% were KOH negative and culture positive Culture was found to be superior to KOH wet mount Though KOH wet mount is rapid, but at times gives false negative results which misleads the clinician Hence all the KOH negative samples need to

be cultured to confirm the diagnosis and to identify the undetected cases The drawback

of fungal culture is that it is time consuming The different clinical types of superficial mycoses and the various fungi isolated in this study are compared with other Indian studies

in table 3, 4, 5, 6 and 7 Onychomycoses (29.17%) was the most common type of superficial mycoses in our study whereas Tinea corporis was the most common type of clinical presentation in other studies (30.19%,

Bhatia et al., and 44.3%, Surendran et al.,) Dermatophytes, especially T.rubrum was the

commonest isolate in most of the studies

(Dulla et al., 2015; Prasad et al., 2013 and Abida Malik et al., 2014) except for the study

done by Bhatia et al., where T mentagrophytes (64.9%) was the most

common dermatophyte isolated Among the

(14.63%) were the most common isolate in our study which was similar to study done by

Surendran et al., (2014) (67.5%)

Table.1 Age distribution of suspected clinical cases

Age group of patients (in years) Number of patients Percentage (%) n=48

Table.2 Frequency of positive and negative results after specimen examinations (Direct

microscopy Vs Culture

Diagnostic test KOH positive KOH negative Total (n=48) Fungal culture positive 37 (77.08%) 4 (8.33%) 41 (85.41%)

Table.3 different types of superficial mycoses cases isolated in our study

Provisional

clinical diagnosis

Number of cases

Percentage of cases (n=48)

KOH mount positive

Fungal Culture positive

Table.4 Comparison of clinical cases isolated with other studies

Provisionalclinical

diagnosis

Present study

Karnataka (Surendran

et al)

Himachal Pradesh (Bhatia

et al)

Table.5 Fungi isolated from superficial fungal infections: A comparison with different studies

conducted in India

Present study

Vijayawada

(Dulla et al)

(Abida Malik et al)

Cuttack study

(Prasad et al)

Table.6 Distribution of Dermatophytic isolates in comparison with other studies

Present study

Dulla et al Bhatia et al Prasad et al Surendran et al Abida Malik et al

Microsporum

gypseum

Table.7 Distribution of Non-dermatophytic fungal isolates in comparison with other studies

Present study

Vijayawada (Dulla.et al)

Karnataka (Surendran et al)

Aligarh (Abida Malik

et al)

Fig.1 Identification of yeasts Culture on SDA

Gram stain of the colony

Germ tube test

Chrom agar Candida

Fig.2 Identification of moulds

Ectothrix

T.rubrum culture Bipolaris in KOH mount (40x)

T rubrum and T verrucosum respectively (LPCB Mount after slide culture)

However, two studies (Dulla et al., 2015 and

Abida Malik et al., 2014) done in Vijayawada

and Aligarh showed that Alternaria (20%)

and A fumigatus (15.6%) were the most

common non-dermatophyte species isolated

respectively From the above comparisons, it

is clear that dermatophytes still continue to be

the commonest causative agents in many parts

of India However, non-dermatophytic molds

are also emerging as important causative

agents and can no longer be neglected The

climatic conditions, overcrowding,

unhygienic habits, occupational conditions

and ignorance of common people have led to

the persistence of these infections even

though many of these infections are easily

treatable

In this study, Bipolaris was the rare fungal

agent to be isolated and not many studies in

India have reported it This suggests that even

rare fungal agents are prevalent in certain

parts of our country and more studies are

required from such regions in this regard

It is concluded that along with dermatophytes,

dermatomycotic fungi are also emerging as an

important cause of superficial mycoses

Though culture was found to be more

sensitive than KOH mount in our study, both

direct microscopy and cultures are important

tools of diagnosis for the superficial fungal

infections

Good hygiene, sanitation and proper hand

washes are effective methods for prevention

of such infections

Acknowledgements

We thank sincerely all the staff members of

department of Dermatology for their timely

support and guidance while conducting this

study

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